1. Field of the Invention
The present invention relates to a method for obtaining information and a device for obtaining information, particularly to a method for obtaining information relating to an object by time-of-flight mass spectrometry. The present invention relates also to a device for obtaining information according to the method.
2. Description of the Related Art
With progress in genome analysis in recent years, analysis of proteins, gene products in living bodies, has become increasingly important. Hitherto, the analysis of a protein formation mechanism and of protein function have been noted and are being developed. Most of the methods of analysis of proteins are based on a combination of the following techniques: (1) isolation and purification by two-dimensional electrophoresis or high-speed liquid chromatography (HPLC), and (2) detection by radiation analysis, optical analysis, mass analysis, or a like analysis method.
The basis of the protein analysis technique is proteome analysis. By this proteome analysis, proteins that are formed by genes and are actually working in a living body are analyzed to investigate functions of cells and causes of diseases. A typical analysis method comprises the following steps: (1) extraction of proteins from an objective biological tissue or cells, (2) isolation of the proteins by two-dimensional electrophoresis, (3) analysis of the proteins or fractions thereof by mass analysis, such as MALDI (matrix-assisted laser desorption)-time-of-flight mass spectrometry (MALDI-TOFMS), and (4) identification of the proteins by utilizing a database, such as a genome project.
Another analysis method comprises the following steps (ISOBE Toshiaki, TAKAHASHI Nobuhiro, Eds. “Experimental Medical Science, additional volume, Proteome Analysis” 2000, Yodosha Co.): (1) extraction of proteins from an objective biological tissue or cells, (2) digestion (or denaturation) of the extracted proteins, (3) analysis of the digested (or denatured) proteins by use of a system that combines liquid chromatography (LC) and ion-trap mass spectrometry (Ion-trap MS), and (4) construction of a database and identification of the proteins.
Such proteome analysis techniques are yielding successful results, for example, in the investigation of the role of a protein in recurrence or metastasis of cancer.
The inventors of the present invention disclosed a method and apparatus for obtaining information on two-dimensional distribution of proteins in a protein chip or a sliced living tissue by visualization using a TOF-SIMS system (time-of-flight secondary ion mass spectroscopy) (Japanese Patent Application Laid-Open No. 2006-10658). In this method, an ionization-promoting substance and/or a digestion enzyme is first applied onto the protein chip or the sliced living tissue by an ink-jet system, and then the information on the kind of protein (including information on the peptides formed by limited decomposition by the digestion enzyme) is visualized by a TOF-SIMS system with the positional information being retained.
Techniques mentioned below are known for analysis of a polypeptide by TOF-SIMS: detection of a polypeptide parent molecule having a large molecular weight by a pretreatment, such as MALDI of mixing a polypeptide and a matrix substance (K. J. Wu et al.: Anal. Chem. 1996, vol. 68, p. 873); detection by imaging a polypeptide by isotope-labeling of a part of a polypeptide with a secondary ion, such as C15N− (A. M. Belu et al.: Anal. Chem. 2001, vol. 73, p. 143); estimation of the kind of a poly-peptide from the kinds of the fragment ions (secondary ions) of the amino acid residues and the relative intensity thereof (D. S. Mantus et al.: Anal. Chem., 1993, vol. 65, p. 1431); investigation of the detection limit of a polypeptide adsorbed on substrates by TOF-SIMS (M. S. Wagner et al.: J. Biomater. Sci. Polymer Edn., 2002, vol. 13, p. 407); and increase of detection sensitivity by chemically modifying a polypeptide with gold nano-particles (Y-P. Kim et al.: Anal. Chem., 2006, vol. 78, p. 1913).
The above-mentioned method for obtaining information disclosed by the inventors of the present invention (Japanese Patent Application Laid-Open No. 2006-10658) provides information on proteins in diseased tissue and normal tissue (including information on a limited decomposition of a peptide by a digestion enzyme). However, it is desirable to improve detection sensitivity in this method. The method disclosed in ISOBE Toshiaki, TAKAHASHI Nobuhiro, Eds. “Experimental Medical Science, additional volume, Proteome Analysis” 2000, Yodosha Co., detects the parent molecule, even a high-molecular polypeptide, with the molecular weight retained by inhibiting the decomposition caused by primary ion radiation. This method uses a mixture of the polypeptide and a matrix substance as the measurement specimen. Therefore, this method cannot provide information on the original two-dimensional distribution in the aforementioned protein chip. The method disclosed by A. M. Belu et al. (Anal. Chem. 2001, vol. 73, p. 143) labels a part of an objective polypeptide with an isotope and detects the polypeptide with a high spatial resolution of TOF-SIMS. However, the isotope-labeling of the objective polypeptide in every measurement is problematic. The method disclosed by D. S. Mantus et al. (Anal. Chem., 1993, vol. 65, p. 1431) estimates the kind of a polypeptide based on the fragment ions (secondary ions) of the amino acid residues and relative intensities thereof. This method cannot discriminate the polypeptides of analogous amino acid constituents in a mixture.
In another method, the sensitivity in parent molecule detection is improved by retarding formation of fragment ions of a polypeptide by use of a metal substrate or metal fine particles. In the method disclosed by M. S. Wagner et al. (J. Biomater. Sci. Polymer Edn., 2002, vol. 13, p. 407), the sensitivity is improved by promoting ionization of a parent polypeptide molecule. Specifically, in this method, a polypeptide is initially placed in a layer that is only several molecules thick in a thin film state on a metal substrate; a primary ion beam is projected through the polypeptide film to impact the substrate; the recoil energy from the substrate dissociates effectively the molecules on the substrate; and the dissociated molecules are allowed to fly upward freely out of the thin film. Thereby, the ionization of the polypeptide parent molecules is promoted by retarding the fragment-ionization of the polypeptide to improve the detection sensitivity. In the method disclosed by Y-P. Kim et al. (Anal. Chem., 2006, vol. 78, p. 1913), the polypeptide molecules are modified respectively at the one end by a gold fine particle and are allowed to orient on a substrate, and a primary ion beam is projected to impact against the gold fine particles in a manner similar to the above-mentioned method of M. S. Wagner et al. (J. Biomater. Sci. Polymer Edn., 2002, vol. 13, p. 407). Thereby, in this method, the molecules on the fine particles are dissociated and allowed to fly out by the recoil energy from the gold atoms to promote ionization of the polypeptide parent molecules and to improve the detection sensitivity. However, these two methods require the step of forming a several-molecule thin polypeptide film or the step of modifying the polypeptide with gold fine particles. Therefore, these two methods cannot provide information on the two-dimensional distribution of the polypeptides in a protein chip or a biological specimen.
Accordingly, for analysis of a protein chip or a biological specimen by TOF-SIMS, improvement is desired for sensitivity in detection of polypeptide parent molecule ions without decomposition into fragments by secondary ions. The improvements disclosed so far are not satisfactory, as discussed above.
The present invention is made to solve the above-noted problems that exist in the prior art and is intended to provide a method for obtaining information for deriving a two-dimensional distribution image with high spatial resolution. Also, the present invention is intended to provide a device for practicing the method for obtaining the information.